Blank Western blot - (Nov/02/2005 )

hi,
I am having troubles with my WBs... they are completly blank. I have tried different primary antibodies and I know that secondary antibodies, ECL reagents, PBS, the flims and the developing machine are working. To be sure that I had proteins on the membrane, I stained it and There are a lot of bands!!
So, Do you have any idea of what can be happened? Can the loading buffer interfere with the antigen-antibody binding in any way?
Thank you

What proteins are you looking at? What was the isloation of said proteins? Length of transfer? Type of membrane?

One thing I would do... (be careful not to let the membrane dry out)
1) prepare the membrane as one would do before transfer (ie. PVDF soaked in methanol )
2) blot the raw protein fraction directly onto the membrane
3) reduce the protein with loading buffer and blot onto the membrane
4) develop the membrane as normal (ie. blocking wash, primary antibody wash ect.)
5) depending on your detection use an statndard so you know the dectection process is working (ie. chemilus. I use magic mark xp as a weight standard)

Results: you should see three spots on the membrane (one non-denatured samples, one denatured sample, one control)
With this test you will know if the transfer process is not working or if the loading buffer is not working, or if the isolation, or primary antibodies are not working

Just because the membrane has proteins when stained, your protein of interest could not be there because of it being modified in some way to hamper transfer.

Good luck

Lou

Are you washing your membrane enough? I wash 6 times (5mins each) with TBS-T after adding the primary then 5 times (5 mins each) with TBS-T and once with PBS.
On the other hand the PhD student who taught me a lot about how to do Westerns spent the last three months she was in the lab getting blank blots so it could just be one of those things!
Have you considered developing an ELISA assay for your protein?

I'd proceed as Cow Guy recommends -- take the whole PAGE and transfer operations out of the equation by doing dot blots on samples at various stages of your purification steps to see if it's a loss of protein or primary antibody problem...

Hi!
Stupid question...
Are you sure you use the appropriate secondary for your primary one?

Good luck.
Cheers

Thank you lou,
These are mitochondrial proteins isolated by differential centrifugation at ph6. The transfer was at 100 V for 1 h 10 minutes and the membrane is PVDF.
I am going to follow your suggestions.
The most funny thing is that I have used as a control another membrane that worked a week ago and now it seems it does not work anymore.

Thank you very much
sela

HI again,
I washed the blots enough and the secondary is OK. One of my labmates is using it and it works for him.. Another student is using my primary antibodies to check them and they are fine.
I am runnning out of answers.
Thank you
Sela

Hey Sela
Just a suggestion. Is it possible that your protein is very low prevalence? If so, you may try with some biotinylated primary antibody, so that you can enhance the final signal. Or you can try loading more amount of total protein. However, you can't use more than 50-60ug of total protein in all as it may create problems with running the PAGE. So be careful. Nothing else comes to my mind as yet. Will think about more options...

AZCBIO

Hi Sela:
That problem has happened in my lab some times, and it is allways related to the blocking solution. Do you use 5% milk? We found that when it gets old , we get problems.
Try to use fresh 5% milk . Even brand NEW.
Tip : keep the powder at 4 C. It would help. You can check with your old blot that worked the first time but not the second. I hope it helps.